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Feb 15, 2011 (Vol. 31, No. 4)

Gene Delivery on a Rebound

As Researchers Correct Past Shortcomings, Promising New Pathways Begin to Appear

  • Cut It Out

    Click Image To Enlarge +
    Cellectis is developing various genome engineering applications using meganucleases. The process involves introducing into the cell a meganuclease that is specific to the targeted site and inserting a gene with the attributes required to stimulate homologous recombination. The meganuclease breaks the DNA molecule and the homologous recombination system corrects this break by taking as a model the gene introduced at the same time as the meganuclease. [© Frank Beloncle]

    Gene therapy can be used against viruses as well. Cellectis Therapeutics (a subsidiary of Cellectis) uses meganucleases engineered to precisely disrupt integrated viruses such as HIV, HBV, and HSV.

    Meganucleases (also known as homing endonucleases) target a specific, statistically unique, sequence—generally at least 14 base pairs—creating a double-strand break in the DNA. The cell then attempts to repair the break, often with some minor mutation or loss of sequence.

    Cellectis’ most advanced meganuclease project targets an essential gene in the herpes simplex virus. “We were able to show that if we take cells and introduce a plasmid that expresses a meganuclease that targets the viral genome, and then we infect those cells with an HSV virus, we can decrease the infectivity of that virus,” related Julianne Smith, Ph.D., head of the meganuclease recombination system group. “The number of infected cells significantly decreases when the meganuclease is present.”

    If there is a homologous sequence present, the cell may attempt to repair the break by homologous recombination. In a therapeutic context, “you could go in and actually create a double-stranded break and introduce a plasmid that contains wild-type information, and go on to correct the mutated allele at its genomic location.”

    To test this idea, Cellectis developed a series of meganucleases targeting the RAG-1 gene (involved in a form of severe combined immunodeficiency), and used them to demonstrate homologous recombination between the chromosomal locus and a repair plasmid.

    The efficiency by which meganucleases get into cells and do their job is about the same as other means of gene therapy. The important difference is the safety aspect, said Dr. Smith. “If we can specifically target a genomic site, we could avoid some of the serious adverse effects that have been observed concerning random insertions and activation of adjacent genes.” Similarly, random insertions are also subject to gene silencing through gene extinction, but with precise targeting you can select your locus.

    In certain cases, it’s even possible to use meganucleases to generate an event in a cell that couldn’t be done any other way, she added, such as cleaving the genome of a virus or perhaps even knocking out a gene. Most antivirals are based on blocking replication, not on eliminating the genome.

  • Image-Guided, Convection-Enhanced

    Precise delivery also is of paramount concern to the University of California, San Francisco’s Krystof Bankiewicz, M.D., Ph.D. He too is worried about causing toxicity as well as engendering adverse immune reactions. Specifically, his work focuses on delivering therapies—whether genetic or pharmaceutical—to specific regions of the brain for the treatment of cancers and neurological disorders.

    Typically the location of an intracerebral injection was imprecise and, because of the pressure in the brain, there was a propensity for therapeutics to travel along the needle and back to the surface. Dr. Bankiewicz’ group developed a delivery system that utilizes MRI to guide, in real-time, a small-diameter silica and ceramic cannula to exactly where the therapy is needed.

    By gently increasing the pressure at the tip of the cannula, the extracellular fluid is pushed away, “allowing us to infuse pretty large volumes of the fluid that find a space now because it displaces fluid that is already in the brain,” he explained. A tracer in the injection allows it to be tracked. “Basically any structure within the brain”—even large volumes of the brain—can be covered at will by viral particles in a “phenomenally” precise and predictable manner.

    It’s not as simple as just watching as a needle goes into the brain, squirting, and watching as the injection magically reaches its target, though. The delivery system itself is like one leg of a three-legged stool. Dr. Bankiewicz has spent years studying things like the movement of cerebrospinal fluid. “It’s being driven by blood vessels causing peristaltic action for the perivascular space.”

    “It’s highly predictable and well described how the fluid circulates within the brain.” It’s also of vital importance to know what the viral vector itself is doing: AAV has multiple serotypes, utilizing different trafficking patterns and with different propensities to transduce different cell types. “We have developed a full understanding of how specific serotypes behave in the brain, and how much transgene—gene product—is being delivered, and where it goes.”

    The main theme is that if you don’t know how to deliver the drug, it is never going to work, Dr. Bankiewicz summarized. “It will be all over the map—it is never going to be consistent. You have to have a technology that is very precise, very predictable, and reproducible.”


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